CNG Channel Gating Movements Monitored via Fluorescene Quenching

描述（由申请人提供）：环核苷酸门控（CNG）离子通道是通过细胞质环核苷酸与保守环核苷酸结合结构域（CNBD）的协同结合调节的变构蛋白。CNG通道产生负责将视觉和嗅觉信息转导到大脑的主要电信号。CNG通道中的人类突变引起各种疾病，包括完全色盲或色盲和视网膜色素变性，其导致视网膜变性和失明。配体结合结构的C-末端结构域的HCN 2，一个密切相关的离子通道，其中包括CNBD，已确定由X-射线晶体学。该结构域由一个八链的“卷”和两个螺旋（B和C）形成，环核苷酸结合在C螺旋和"卷“之间的口袋中。未配体的CNBD的结构和伴随配体结合和通道门控的运动的细节尚未确定。所提出的研究的目标是使用溶液和完整通道中分离的CNBD的荧光测量来监测这种构象变化。连接到通道上的各个位置的荧光团和猝灭剂之间的距离依赖性猝灭将解决关于CNBD构象变化的性质的具体问题。在激动剂结合和通道门控时，C螺旋如何相对于卷重定向？在没有激动剂的情况下，C螺旋是否保持其二级结构？荧光测量可以与CNG通道的电流测量同时进行，这允许结构变化与功能蛋白质状态直接相关，这是其他类型蛋白质不可能的分辨率。因此，所提出的研究不仅将提供关于离子通道门控机制的信息，而且还将提供对结构相关分子（包括某些激酶、转录因子和鸟嘌呤核苷酸交换因子）运动的深入了解。公共卫生相关性本研究的目的是了解在一个称为环核苷酸门控离子通道的蛋白质家族中发生的动态结构重排。这些蛋白质是将视觉信息从眼睛传递到大脑所必需的，编码这些通道的基因突变会导致失明。使用结构和功能测定的组合，这些通道的正常分子运动将被确定，这将有助于理解它们在健康个体中通过细胞信号的调节，以及它们在疾病中的失调。

DESCRIPTION (provided by applicant): Cyclic nucleotide-gated (CNG) ion channels are allosteric proteins regulated by the cooperative binding of cytoplasmic cyclic nucleotides to a conserved cyclic nucleotide binding domain (CNBD). CNG channels generate the primary electrical signal responsible for transduction of visual and olfactory information to the brain. Human mutations in CNG channels cause various diseases including complete achromatopsia or colorblindness and retinitis pigmentosa, which leads to retinal degeneration and blindness. The ligand-bound structure of the C-terminal domain of HCN2, a closely related ion channel, which includes a CNBD, has been determined by x-ray crystallography. This domain is formed by an eight-stranded ¿ roll followed by two helices (B and C) with cyclic nucleotide binding in a pocket between the C helix and the ¿ roll. The structure of the unliganded CNBD and the details of the movements accompanying ligand binding and channel gating have not been determined. The goal of the proposed study is to monitor this conformational change using fluorescence measurements of isolated CNBD's in solution and intact channels. Distance-dependent quenching between fluorophores and quenchers attached to various positions on the channel will address specific questions about the nature of the CNBD conformational change. How does the C helix reorient relative to the ¿ roll upon agonist binding and channel gating? Does the C helix maintain its secondary structure in the absence of agonist? Fluorescence measurements can be performed simultaneously with current measurements from CNG channels, which allows for structural changes to be correlated directly with a functional protein state, a resolution not possible for other types of proteins. Therefore, not only will the proposed studies be informative with respect to ion channel gating mechanisms, but they will also provide insight into the movements of structurally-related molecules, including certain kinases, transcription factors and guanine nucleotide exchange factors. PUBLIC HEALTH RELEVANCE The goal of this study is to understand the dynamic structural rearrangements that occur in a family of proteins known as cyclic nucleotide-gated ion channels. These proteins are necessary to relay visual information from the eye to the brain, and mutations in the genes encoding these channels result in blindness. Using a combination of structural and functional assays, the normal, molecular motions of these channels will be determined, which will be instrumental in understanding their regulation by cellular signals in healthy individuals, and their misregulation in disease.